Equine herpesvirus 1 (EHV-1), a member of the Alphaherpesvirinae, is the major cause of virus-induced abortion in equids and causes respiratory and neurological disease. The entire DNA sequence of the EHV-1 strain Ab4p has been determined (Telford, E. A. R. et al., 1992). Only few genes and gene products have been characterized for their relevance for the virulence or immunogenicity of EHV-1 because the production of viral mutants is still relying on the generation of recombinant viruses by homologous recombination between the viral genome and respective foreign DNA to be inserted in cultured mammalian cells.
For control of EHV-1 infections, two different approaches are followed. First, modified live vaccines (MLVs) have been developed, including the strain RacH (Mayr, A. et al., 1968; Hübert, P. H. et al., 1996), which is widely used in Europe and the United States. Second, inactivated vaccines and independently expressed viral glycoproteins have been assessed for their immunogenic and protective potential. Among the glycoproteins that were expressed using recombinant baculoviruses are the glycoproteins (g) B, C, D, and H, which induced partial protection against subsequent challenge EHV-1 infection in a murine model (Awan, A. R. et al:, 1990; Tewari, D. et al., 1994; Osterrieder, N. et al., 1995; Stokes, A. et al., 1996). However, the use of MLVs has advantages over killed and subunit vaccines. MLVs are highly efficient in inducing cell-mediated immune responses, which are most likely to be responsible for protection against disease (Allen, G. P. et al., 1995; Mumford, J. A. et al., 1995).
Herpesvirus glycoproteins are crucially involved in the early stages of infection, in the release of virions from cells, and in the direct cell-to-cell spread of virions by fusion of neighboring cells. To date, 11 herpes simplex virus type 1 (HSV-1)-encoded glycoproteins have been identified and have been designated gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL, and gM. HSV-1 mutants lacking gC, gE, gG, gI, gJ, and gM are viable, indicating that these genes are dispensable for replication in cultured cells. Comparison of the HSV-1 and equine herpesvirus 1 nucleotide sequences revealed that all of the known HSV-1 glycoproteins are conserved in EHV-1. According to the current nomenclature, these glycoproteins are designated by the names of their HSV-1 homologs. In addition, a further envelope protein named gp1/2 and a tegument protein, the VP13/14 homolog of HSV-1, have been described to be glycosylated in case of EHV-1 (reviewed in Osterrieder et al., 1998). It is known that EHV-1 gC, gE gI, and gM are not essential for growth in cell culture, whereas gB and gD appear to be essential for virus growth in cultured cells. The contributions of other EHV-1 glycoproteins to replication in cultured cells are not known (Neubauer et al., 1997; Flowers et al., 1992).
The gp1/2 glycoprotein is encoded by gene 71 (Wellington et al., 1996; Telford et al., 1992) and was also shown to be nonessential for virus growth in vitro (Sun et al., 1996). In addition, a viral mutant carrying a lacZ insertion in the gene 71 open reading frame was apathogenic in a murine model of infection but still able to prevent against subsequent challenge infection (Sun et al., 1996; Marahall et al. 1997). In addition, the KyA strain of EHV-1 harbors a major deletion in the coding sequences of gene 71 (Colle et al., 1996).
The technical problem underlying this invention was to provide a new tool and procedure to generate attenuated equine herpesviruses of defined specificity.